Metal Abstraction Peptide (MAP) Tag and Associated Methods

ABSTRACT

Compositions comprising a tripeptide having the sequence XC 1 C 2 ; wherein X is any amino acid such that XC 1 C 2  is capable of binding a metal in a square planar orientation or square pyramidal orientation or both; and wherein C 1  and C 2  are the same or different; and wherein C 1  and C 2  individually are chosen from a cysteine and a cysteine-like nonnatural amino acid, as well as metal-XC 1 C 2  complexes and methods for forming such complexes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation under 35 U.S.C. §120 of U.S. Ser. No.13/341,223, filed Dec. 30, 2011, which is a continuation under 35 U.S.C.§120 of U.S. Ser. No. 12/465,448, filed May 13, 2009, now U.S. Pat. No.8,110,402, issued on Feb. 7, 2012, which claims priority under 35 U.S.C.119(e) to U.S. Provisional Application 61/052,918 filed on May 13, 2008,of which all of the above are incorporated by reference in theirentirety.

STATEMENT OF GOVERNMENT INTEREST

The present invention was developed under a grant from NationalInstitutes of Health (Grant No. P20 RR-17708). The U.S. Government hascertain rights to the invention.

SEQUENCE LISTING

This disclosure includes a sequence listing submitted as a text filepursuant to 37 C.F.R. §1.52(e)(v) named 41861701305SeqListing.txt,created on Aug. 28, 2012, with a size of 2.4 kb, which is incorporatedherein by reference.

BACKGROUND

Previously, it has been a common technique to utilize metals to extractpolypeptides (e.g., proteins or protein fragments) from compositions.Such extraction has been based on the ability of a metal to complex witha specific polypeptide over other substances within the composition. Itis known that two different peptide-based metal binding tags can be usedfor separating a peptide from a composition. However, these binding tagshave specific types of amino acids, and specific numbers or specificsequences of amino acids that are utilized to accomplish binding thatare substantially different from those described in connection with thepresent invention. For example, these two binding tags may not bind thesame types of metals or bind using the same geometry as the presentinvention. Additional information regarding the first binding tag can beobtained in U.S. Pat. No. 7,208,138 B2 by Haroon et al., and pertains toa peptide comprising the sequence NXEQVSP (SEQ ID No: 7). Informationregarding the second binding tag can be obtained in U.S. patentapplication publication 2004/0018974 by Arbogast, et al., and pertainsto a sequence of the tag that appears to be the entire protein describedtherein.

Tags that can be encoded in the genetic material of an organism forrecombinant expression of proteins have been utilized extensively forpurification and identification of protein products. The most noteworthyexample of this has been the His-tag technology, which provides a facilemeans of effectively isolating the tagged protein from whole cells usingimmobilized metal affinity chromatography (IMAC). Numerous otherpeptide-based tags have been developed for detecting a tagged protein incell culture assays or cell lysates using antibodies that recognize thepeptide tag. These technologies are useful in in situ or in vitroassays, but they generally are not applicable to in vivo analysis. Theadvantage of a peptide tag is that the tag is covalently attached to theprotein of interest without the need for additional chemical steps tolabel the protein.

MRI imaging is a common method for examining structural features in liveanimals and humans. The technology is safe and non-invasive. Contrastagents have been developed to improve the sensitivity of the method, andthey are used to enhance the features observed in the MRI image. Theimprovement is achieved because contrast agents contain metals thatalter the signal from neighboring molecules, typically water. The mostcommonly used agents chelate Gd(III), but other metals can be used toenhance contrast. Gd-containing contrast agents cannot be used inpatients with compromised kidney function, as serious complicationsknown as Nephrogenic Systemic Fibrosis or Nephrogenic FibrosingDermopathy can result. Alternative imaging agents are needed to addressthe needs of such patients. Compounds that chelate metals are also usedin PET and SPECT imaging to view molecular level details.

SUMMARY

The present disclosure generally relates to tripeptide motifs andmethods of using such motifs. These peptides have the ability to bind tometals, which makes them useful for a variety of applications. Inparticular, the tripeptides of the present disclosure have applicationsin imaging, research, chemotherapy, and chelation therapies.

According to certain embodiments, the present disclosure providescompositions comprising a tripeptide having the sequence XC₁C₂; whereinX is any amino acid such that XC₁C₂ is capable of binding a metal in asquare planar orientation or square pyramidal orientation or both; andwherein C₁ and C₂ are the same or different; and wherein C₁ and C₂individually are chosen from a cysteine and a cysteine-like nonnaturalamino acid.

According to other embodiments, the present disclosure providescompositions comprising a tripeptide having the sequence XC₁C₂ and ametal; wherein the metal is complexed with the tripeptide; and wherein Xis any amino acid such that tripeptide and metal form a complex having asquare planar orientation or square pyramidal orientation or both; andwherein C₁ and C₂ are the same or different; and wherein C₁ and C₂individually are chosen from a cysteine and a cysteine-like nonnaturalamino acid.

According to other embodiments, the present disclosure provides methodscomprising complexing with a metal a tripeptide having the sequenceXC₁C₂ to form a metal-XC₁C₂ complex; wherein X is any amino acid suchthat metal-XC₁C₂ complex has a square planar orientation or squarepyramidal orientation or both; and wherein C₁ and C₂ are the same ordifferent; and wherein C₁ and C₂ individually are chosen from a cysteineand a cysteine-like nonnatural amino acid.

The features and advantages of the present invention will be readilyapparent to those skilled in the art upon a reading of the descriptionof the embodiments that follows.

DRAWINGS

A more complete understanding of this disclosure may be acquired byreferring to the following description taken in combination with theaccompanying figures.

FIG. 1 shows Structure 1—a GGH-like metal complex NGH, with 4Ncoordination. Structure 2 depicts a GGH-like metal coordination by a NCCpeptide, whereas Structure 3 depicts the unique, high-affinity bindingarrangement of NCC, involving 2N:2S coordination.

FIG. 2 shows a sequence alignment of the C-terminal portion of human PRLenzymes, including PR1-1, PRL-2, and PRL-3. H166 of PRL-1 is part of theNGH motif shown underlined. Residues C170 and C171 of PRL-1 are part ofthe CaaX motif shown in italics. The NCC motif is bold.

FIG. 3 shows A. ESI-MS of the nickel-bound tripeptide acquired at pH 10,where the mass indicates that nickel is bound to the peptide. B. ESI-MSspectrum after dropping the pH of the sample to 5, showing that nickelhas been released from the peptide.

FIG. 4 shows Cu-NCC magnetic resonance image (MRI).

FIG. 5 shows a photograph of purified WT PRL-1 purified in the presenceof Ni(II), pH 7.4 after cleavage and removal of the His-tag. Photographshows its characteristic rust color.

FIG. 6 shows A) Electronic absorption spectra of PRL-1 variants purifiedin the presence of Ni²⁺ ion. Three (two) spectra for each proteinvariant were recorded from 800 to 200 nm and averaged. Proteinconcentration for all variants is 5 mg/mL to within 10% based on A280comparison. Shown are the spectra from 700 to 300 nm. Visible featuresare absorbance maxima at 318 and 421 nm. Inset. The spectra from 600 to400 nm are shown at smaller scale for visualization of absorption maximaat 421 and 526 nm. Curves are as follows: PRL-1-WT, black (top);PRL-1-H166A, red (next to top); PRL-1-C170S-C171S, orange (bottom);PRL-1-C1705, green (next to bottom); PRL-1-C171S, blue (middle). B)Resolved spectrum of PRL-1 WT. Resolution of the UV-V is spectrum ofNi-purified PRL-1 WT protein between 300 and 700 nm fits the data using5 curves. The 318 nm shoulder is resolved into peaks at 306, 325, and372 nm. The sum of the resolved absorbance peaks (gray dashed line) andthe raw data (solid black line) are both shown. The peaks at 421 and 526nm are also present.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent Office upon request andpayment of the necessary fee.

While the present disclosure is susceptible to various modifications andalternative forms, specific example embodiments have been shown in thefigures and are herein described in more detail. It should beunderstood, however, that the description of specific exampleembodiments is not intended to limit the invention to the particularforms disclosed, but on the contrary, this disclosure is to cover allmodifications and equivalents as defined by the appended claims.

DESCRIPTION

The present invention generally relates to short peptide motifs andmethods of using such motifs. These peptides have the ability toselectively bind to metals, which makes them useful for a variety ofapplications. In particular, the peptides of the present invention haveapplications in imaging, research, chemotherapy, and chelationtherapies.

Generally, the present disclosure is related to short, novel peptidemotifs that strongly bind with a select metal, referred to as MAPtag(s). As such, these MAP tags can be used, among other things, toextract the select metal from a composition. The MAP tags of the presentdisclosure are 3 amino acids in length, and may be included in longerpolypeptides and proteins at the N-terminus, C-terminus, or any positionin between. In certain embodiments, however, it may be advantageous forthe MAP tag to be present in a polypeptide or protein configuration thatpresents the MAP tag for binding with a metal, such as being present inan external loop. The MAP tag also may be attached to a non-peptideentity. Additionally, more than one MAP tag may be present on aparticular molecule.

It is known that many proteins bind metals. However, such bindingtypically utilizes discontinuous sequences of amino acids within afolded protein structure to accomplish tight binding, which uponunfolding then easily release the metal. On the other hand, metalbinding by MAP tags is accomplished using atoms in very close proximity,and as such, extreme conditions are required to release the metal.Thermal and chemical denaturation of the protein permits slow release ofthe metal. For example, use of extreme conditions (e.g. boilingtemperature, strong acid) may lead to slow release of the metal over aperiod of time (e.g., several to many hours).

A. Chemical Structure

In certain embodiments, the present disclosure provides a novelpeptide-based tag or binding agent for a metal (a MAP tag). The MAP tagcan be used to directly encode a high-affinity metal binding site inpolypeptides or proteins. The MAP tags of the present disclosure arecharacterized by their ability to bind metals in a square planar and/orsquare pyramidal geometry.

1. Peptide Sequence

The MAP tags of the present disclosure generally comprise at least threecontiguous amino acid residues capable of binding a metal. The MAP tagsof the present disclosure generally have a sequence represented byXC₁C₂, in which C₁ and C₂ may be the same or different and may be acysteine, or a cysteine-like nonnatural amino acid (e.g., a sulfurcontaining alpha- or beta-amino acid), and in which X may be anothernatural or nonnatural amino acid or amino acid analogue, so long as thepeptide tag formed is capable of binding a metal in squareplanar/pyramidal geometry. As used herein, the abbreviations for theL-enantiomeric amino acids are conventional and are as follows:

TABLE 1 Abbreviations of the natural amino acids. One letter Threeletter Amino acid symbol symbol alanine A Ala arginine R Arg asparagineN Asn aspartic acid D Asp cysteine C Cys glutamic acid E Glu glutamine QGln glycine G Gly histidine H His isoleucine I Ile leucine L Leu lysineK Lys methionine M Met phenylalanine F Phe proline P Pro serine S Serthreonine T Thr tryptophan W Trp tyrosine Y Tyr valine V ValIn some embodiments, X may be asparagine (N), glutamine (Q), histidine(H), lysine (K), or arginine (R).

When an amino acid sequence is represented as a series of three-letteror one-letter amino acid abbreviations, it will be understood that theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy terminal direction, in accordance with standardusage and convention.

The MAP tag of the present disclosure can be encoded in line with a geneor nucleotide sequence for expression using any recombinant technologysystem. Additionally, it can be incorporated into a peptide or proteinusing any synthetic or biosynthetic method for peptide or proteinproduction. In application, the MAP tag spontaneously reacts with ametal to form a peptide-metal complex. Such peptide-metal compelxes mayform in solution or via transmetallation or any other process.

The MAP tag can be used alone or as a tag that can be attached to theN-terminus, C-terminus or inserted into a flexible linker or loop withina polypeptide chain. The MAP tag can be modified at any position otherthan at the side chains involved in metal coordination. Accordingly, theMAP tag may comprise sequences such as Z—XC₁C₂—Z¹, Z may be any aminoacid or any sequence of amino acids, and Z¹ may be any amino acid orsequence of amino acids that is equivalent or not equivalent to Z. Theamino acids are represented as in above Table 1. Non-natural and aminoacids analogues are not listed in the table, but they may beincorporated into Z, Z¹, and/or X.

In certain embodiments, the MAP tag may be attached to another molecule.For example, the MAP tag may be attached to a non-peptide entity like acarbohydrate (e.g., hyaluronic acid). The attachment may be covalent,and may be affected through a linker.

In some embodiments, the MAP tag may comprise a sequence as follows:NC₁C₂; Z—NC₁C₂—Z¹; Z—NC₁C₂; NC₁C₂—Z¹; QC₁C₂; Z-QC₁C₂—Z¹; Z-QC₁C₂;QC₁C₂—Z¹; HC₁C₂; Z—HC₁C₂—Z¹; Z—HC₁C₂; HC₁C₂—Z¹; KC₁C₂; Z—KC₁C₂—Z¹;Z—KC₁C₂; KC₁C₂—Z¹; RC₁C₂; Z—RC₁C₂—Z¹; Z—RC₁C₂; or RC₁C₂—Z¹. As above, Zmay be any amino acid or any sequence of amino acids, and Z′ may be anyamino acid or sequence of amino acids that is equivalent or notequivalent to Z. The amino acids are represented as in above Table 1.Non-natural and amino acids analogues are not listed in the table, butthey may be incorporated into compounds Z and Z¹.

In one embodiment, the MAP tag comprises NCC having the followingstructural formula:

In certain embodiments, a MAP tag of the present disclosure may beencoded in line with a gene or nucleotide sequence that provides fortargeted delivery of the MAP tag, either before MAP tag complexationwith a metal or after complexation with a metal. This may beaccomplished using genes, peptides, or other motifis known to be usefulfor targeting. For example, MAP tags may be incorporated with anantibody, growth factors, peptides, and the like.

2. Metal Binding

In certain embodiments, the MAP tags of the present disclosure, alone orwhen incorporated into a polypeptide or protein, may complex with ametal to form a MAP tag-metal complex having a square planar/pyramidalgeometry. The metal may complex with the MAP tag through 2N:2Scoordination. The MAP tags also may bind a number of metals in a squareplanar geometry under suitable conditions that will be appreciated byone of ordinary skill in the art. In general, the MAP tags of thepresent disclosure may bind, referring to IUPAC Group: Group 3 metals,such as Y; Group 5 metals, such as V and U; Group 6 metals, such as Cr,Mo, W; Group 7 metals such as Mn, Tc, Re; Group 8 metals, such as Fe andRu; Group 9 metals, such as Co, Rh, Ir; Group 10 metals such as Ni, Pd,Pt; Group 11 metals, such as Cu, Ag, Au; Group 12 metals, such as Zn,Cd, Hg; Group 13 metals, such as Al, Ga, In, Tl; Group 14 metals, suchas Sn and Pb; and Group 15 metals, such as Bi. In certain embodiments,the MAP tag may bind and form a MAP tag-metal complex with Zn, Ni, CuPt, Pd, Au, Ag, Pb, and Fe.

As mentioned above, upon binding the metal, the MAP tag has a squareplanar geometry. The present disclosure also contemplates MAP tag metalcoordination in a variety of square planar configurations. For example,when the MAP tag is NCC and the metal is any metal as described above,and represented as M, the MAP tag may have a structural formularepresented by Formula 1:

Other structural configurations are also contemplated. For example, whenthe MAP tag is NC₁C₂ and C₁ is a nonnatural amino acid, such as(S)-2-amino-2-mercaptoacetic acid and C₂ is cysteine, the MAP tag'sstructural formula may be represented as Formula 2, in which M is anymetal as described above:

In another example, the MAP tag is NC₁C₂ and C₂ is a nonnatural aminoacid, such as (S)-2-amino-4-mercaptobutanoic acid, the MAP tag'sstructural formula may be represented as Formula 3, in which M is anymetal as described above:

In another example, the MAP tag is NC₁C₂ and C₂ is a β amino acid, suchas 3-amino-2-mercaptopropanoic acid, the MAP tag's structural formulamay be represented as Formula 4, in which M is any metal as describedabove:

In another example, the MAP tag is RCC and the MAP tag's structuralformula may be represented as Formula 5, in which M is any metal asdescribed above:

In another example, the MAP tag is QCC and the MAP tag's structuralformula may be represented as Formula 6, in which M is any metal asdescribed above:

B. Applications of the MAP Tag

1. MAP Tags for Metal Abstraction

As mentioned above, a MAP tag is capable of binding metals with highaffinity. A MAP tag is also capable of abstracting a metal from variouscompositions ranging from fluids to solids. Consequently, the ability ofMAP tags to abstract the metal, rather than share coordination, makethem amenable for use in separating a specific metal from anothercomposition, as described more fully below. The MAP tag sequesters ametal ion from compositions by complexing with the metal and thenabstracting or removing the metal from a component in the composition,such as a chelating agent (e.g., EDTA) or a solid support conjugatedwith, for example, IDA or NTA. As such, the MAP tag is a metalabstraction peptide (MAP) tag.

Additionally, due at least in part, to a MAP tag's ability toselectively bind to metals, embodiments of the present disclosure may beuseful for chelation therapy. Thus, a MAP tag may be used to sequester abiologically toxic metal, such as lead.

Similarly, MAP tags also may be useful in sequestering metals fromwater, other aqueous solutions, or any compositions outside of the body.The MAP tag itself is generally soluble in numerous solvents. However,in certain embodiments, the MAP tag-metal complex is poorly soluble inmany organic solvents and may precipitate once metal binding occurs.This allows for efficient separation during the extraction from anorganic solvent.

2. Imaging Applications

In certain embodiments, the present disclosure provides methods forimaging using a MAP tag. Such methods may be used for therapeutic andmedical imaging, or for other imaging applications. The metal chosen tobe complexed with the MAP tag will depend on the particular applicationand imaging technique. For examples, a paramagnetic metal may be chosenfor use with imaging techniques such as MRI, NMR and EPR, while gold maybe chosen for techniques such as electron microscopy, while zinc may bechosen for applications such as fluorescence. Which metals are suitablefor a given application and imaging technique are well-known to thoseskilled in the art.

In one embodiment, a MAP tag is utilized to prepare a reagent for use inimaging techniques. That is, a MAP tag is bound to a metal, which inturn provides a complex that can be used for imaging (e.g., used in abody of a subject such that the metal can be use for imaging). Examplesof suitable imaging techniques include, alone or in combination,Computed Tomography (CT), Magnetic Resonance Imaging (MRI), ultrasound,Positron Emission Tomography (PET), Single Photon Emission ComputedTomography (SPECT), Nuclear Magnetic Resonance (NMR), ElectronParamagnetic Resonance (EPR), electron microscopy, fluorescence imaging,and the like.

In another embodiment, a MAP tag may be incorporated into a polypeptideor protein, or non-protein entity that localizes to a portion of asubject, where the metal portion of a MAP tag-metal complex provides theability to image the site of localization, such as a tissue or tumor.For example, the MAP tag-metal complex can be used to identify a tissueor other structure expressing a receptor and/or binding partner to thetargeting portion of the MAP-tagged molecule. Imaging techniques thatcan visualize the metal can be used for the identification of suchfeatures.

For example, MRI contrast agents are paramagnetic species, typicallycontaining a metal center, that alter the relaxation of neighboringatoms to generate a difference in the detected signal between the areaswhere the contrast agent is present and those where it is not. A MAP tagmay be paramagnetic when complexed with Cu. A MAP tag-metal complextherefore has potential for use as a MRI contrast agent. Most contrastagents are not targeted on a microscopic level but are used to revealmacroscopic structures and defects in a body or tissue. In this type ofapplication, the concentration of the contrast agent is very high.Targeting a contrast agent to a selected tissue or microscopic featureis beneficial in that it permits identification of aberrations on thecellular level. For example, the MAP tag may be expressed as a tag on anantibody that recognizes cancer cells expressing a specific receptor.Targeted delivery may allow for a lower concentration of contrast agentto be administered, which, among other things, may help minimizetoxicity and other side effects.

It it known that ⁶²Cu is a radioactive isotope that is used in PET andSPECT imaging, which are capable of imaging molecular details within abody. As such, a MAP tag can be loaded with the appropriate radioisotopeof Cu (or another metal) and used as a tracer with these imagingtechniques. Similarly, by fusing the tag to any protein or peptide orother molecule that binds a subset of cells, targeted imaging can beaccomplished.

The MAP tag is advantageous, among other things, because it is a shortsequence with high affinity for certain metals. Thus, by encoding a MAPtag in an expressed protein or other molecule, targeted delivery to aspecific cell type within an organism is possible. Further, targeting asubset of cells permits a decrease in the amount of contrast agentneeded for imaging.

3. Chemotherapy

The present invention may also be used to aid in chemotherapy. Forexample, a MAP tag may form a complex with platinum and/orradionucleotides and be used to provide targeted delivery to aparticular cell or type of cell.

To facilitate a better understanding of the present invention, thefollowing examples of specific embodiments are given. In no way shouldthe following examples be read to limit, or to define, the entire scopeof the invention.

EXAMPLES Example 1

Mutagenesis techniques were used to identify the peptide motif havingthe strong binding affinity with select metals. Briefly, the human PRL-1gene was cloned into pET-30 Xa LIC vector (Novagen) and mutated usingthe PCR-based QuikChange method (Stratagene). The primers generating theC170S, C171S, and H166A mutants, 5′ to 3′, wereggtcatagaaacaactCttgcattcaataaggatc (SEQ ID No: 1),ggtcatagaaacaactgttCcattcaataaggctgtaactc (SEQ ID No: 2), andcgtttcaaacattccaacggtGCtagaaacaactgttgcattc (SEQ ID No: 3) (IntegratedDNA Technologies, Coralville, Iowa), respectively. Capital lettersindicate mutated bases. PCR reactions were treated with DpnI (Promega)for 1.5 h at 37° C., directly transformed into NovaBlue GigaSinglesCompetent cells (Novagen) and plated on LB with 30 mg/mL kanamycinselection. Individual colonies were grown overnight in selective LB at37° C. The resultant DNA was purified using the Wizard Plus MiniPrepSystem (Promega). All mutations were confirmed by DNA dye-terminatorsequencing (Northwoods DNA, Inc., Bemidji, Minn.). The double mutantC170S/C171S was produced by a second round of mutagenesis and similarlyconfirmed.

Vectors containing PRL-1 and relevant mutant genes were transformed intoBL21(DE3) cells (Novagen) and grown overnight at 37° C. on selective LB.Selected colonies were grown in selective M9ZB broth overnight at 37° C.Overnight cultures were then transferred to 500 mL unlabeled minimalmedium and induced with 1 mM IPTG at an OD₅₅₀ between 0.6 and 0.8.Proteins expressed for NMR experiments were grown in minimal mediumcontaining chloride. Over-expression of the recombinant protein wasconfirmed by SDS-PAGE. Cells were pelleted by centrifugation at 4400×gand stored at −80° C. until used.

A series of PRL-1 mutants were examined to identify residues thatparticipate in metal coordination. Model peptides containing a GGH-likemotif have been shown to coordinate Ni(II) and Cu(II) with extremelyhigh affinity. In this motif substitution at either of the two Glypositions only modestly decreases binding, whereas mutation of the Hisabolishes it. PRL-1 encodes NGH, a GGH-like consensus motif, near itsC-terminus (FIG. 2). Therefore, PRL-1-H166A was made to test thehypothesis that the NGH sequence contained in PRL-1 is responsible formetal binding. PRL-1 and PRL-1-H166A analyzed by ICP-MS in parallelbound approximately 120 and 60 μM Ni, respectively. Only a 20% decreasein signal was observed for H166A when measured by absorptionspectroscopy. It also appears from the absorption spectra that theprotein signal is diminished to an equivalent extent and that the lossof signal in the visible region is due to protein instability. Proteininstability likely accounts for the disparity in the two detectionmethods since ICP-MS requires substantially more manipulation of thesample prior to analysis. Because the histidine mutant retained tightbinding, mutations at C170 and/or C171 were made because thiols oftenparticipate in Ni coordination. Ni levels in individual cysteinemutants, as well as the double mutant, were greatly reduced relative towild-type protein, indicating both Cys residues influence metalcoordination. No Ni was detected in the C170S mutant or the C170S-C171Sdouble mutant, but a small signal of approximately 17 μM was observedfor PRL-1-C171S.

Example 2

Protein purification techniques were used to purify the protein-metalcomplex. Briefly, cell pellets were resuspended in 30 mL buffer A (100mM NaCl, 50 mM Tris-HCl, 10 mM imidazole, pH 7.4, Ar-sparged) and lysedat 15,000 psi using a French pressure cell (ThermoElectron). Argonsparging of buffers was performed to displace oxygen. Samples then werecentrifuged at 21,000×g and the supernatants were filtered through a 0.2μm nylon filter. Purification of (His)₆-tagged PRL-1 was carried outusing an Äkta Explorer purification system at a flow-rate of 1 mL/min.Samples were applied to a metal-charged 5 ml HiTrap Chelating Column (GEHealthcare) and rinsed with 5 column volumes after loading. Elution wasaccomplished using a linear gradient to 60% buffer B (100 mM NaCl, 50 mMTris-HCl, 500 mM imidazole, pH 7.4, with or without argon sparging) over13 column volumes, to 100% B over 3 column volumes, and maintained at100% B for 4 column volumes. Elution was monitored by absorption at 280nm, and fractions were examined for purity using SDS-PAGE. Theappropriate fractions were pooled and dialyzed against Ar-sparged or notsparged 100 mM NaCl, 50 mM Tris-HCl, pH 7.4. The (His)₆-tag was cleavedfrom the target protein using Factor Xa protease (Promega) overnight atroom temperature. Protease was removed using Xarrest™ agarose (Novagen)and filtered with a 0.2 μm nylon filter before reapplication to thecolumn to remove the tag and any uncleaved protein. The protein wasapplied to the column in dialysis buffer and the initial flow-throughwas collected to make the protein sample. Samples were concentrated andexchanged at least 10⁶-fold in Ar-sparged 100 mM NaCl, 50 mM TrisCl, pH7.4 using (Amicon Ultra) 10 kDa MWCO centrifugal filters. Purity andmutant verification of the samples were ascertained by SDS-PAGE andESI-MS, respectively. Columns were treated with 1 M NaOH, stripped with2 column volumes of 100 mM EDTA and recharged with Ar-sparged 100 mMNiSO₄, 100 mM CuSO₄, 100 mM CuCl₂ or 100 mM ZnCl₂ immediately precedingeach use.

FIG. 5 shows purified PRL-1 after cleavage and removal of the His-tag.Metal bound to the his-tag has octahedral geometry, which is blue-greenin color and the absorption corresponding to such a coordination eventwould appear in the 700-800 nm region of the spectrum. Photograph of WTPRL-1 purified in the presence of Ni(II), pH 7.4 shows itscharacteristic rust color. The purified wild-type PRL-1 protein has anunexpected rust color (FIG. 5), which could result from metalcomplexation or oxidation of aromatic residues (i.e., Trp or Tyr).

Oxidation of aromatic residues was easily ruled out based on the massspectrometry data from the intact and tryptic digest studies of theprotein. Moreover, we observed the density of the protein to be higherthan expected, as the rust-colored protein becomes concentrated at thebottom of the tube during high-speed centrifugation. As metalcoordination would increase the density of the protein complex, wefurther investigated the possibility that the chromophore is generatedby the formation of a metal complex with the protein. Because metalchelation chromatography was used to purify PRL-1, the most likelysource of metal ions was Ni from the IMAC resin.

When cell lysate containing recombinant WT PRL-1 is equally divided andpurified side-by-side using Ni²⁺-, Cu²⁺- or Zn²⁺-charged IMAC resin, theprotein abstracts the metal bound to the chelating resin. The rate oftransmetallation is most rapid with nickel, followed by copper. Zinc ispresent to some extent in all samples, indicating the protein picks upzinc in vivo. When zinc is bound to the column, only zinc is observed inthe protein. Apo PRL-1 is poorly soluble and loss of metal leads toprecipitation. The soluble material from each purification when testedby inductively coupled plasma mass spectrometry (ICP-MS) has anequivalent proportion of metal bound.

Example 3

ICP-MS was used to characterize the bound metal. Briefly, purifiedprotein samples were digested in concentrated nitric acid in PFAmicrocentrifuge tubes (Savillex, #s 7240, 7241) at 68±1° C. for 16.5±0.7hours. Based on protein quantification prior to digestion, the samplewas then diluted to 1.5 μM in 20 mM ammonium bicarbonate, pH 8.2,filtered through a 0.2 μm nylon filter, and injected into a VG ElementalVGII+XS Inductively-Coupled Plasma-Mass Spectrometer fitted with amicroconcentric nebulizer. A minimum of two separately prepared sampleswere analyzed for each protein variant. Each sample was scanned twiceand standards were run before, between, and after each sample scan.Drift was monitored during washes between scans of both sample andstandard injections.

To investigate our hypothesis that the chromophore arises from proteinabstraction of the metal from the charged IMAC matrix, samples weresubjected to ICP-MS for semi-quantitative analysis. As extensive washingis used to remove background metals, the values obtained from thismethod inherently underestimate the amount of metal bound, but can beused to assess the relative affinity of different metals for theprotein. Additionally, to identify an endogenous metal ligand that mayhave been bound to PRL-1 during expression in the cells, ICP-MS was usedto look for the presence of a transition metal from the first two rowsin the periodic table as well as other common biologically relevantmetals. Strikingly, the data showed that PRL-1 bound only to Zn and thespecific metal, for example Ni, used on the IMAC column duringpurification. On average 500 μM Ni-purified protein samples weredigested in nitric acid for 16 h at 70° C. were determined to containapproximately 180 μM Ni, yielding a Ni to protein stoichiometry of1:2.7+/−0.7. Zn was also detected in the Ni-purified samples atapproximately 60 μM. Separately, purification of PRL-1 using Zn-IMACshowed Zn was the only metal present in high quantity at 270 μM [1Zn:1.8 protein], and these protein samples contained only backgroundlevels of Ni. ICP-MS of both Ni- and Zn-purified PRL-1 show no othermetals in significant quantity, which suggests that zinc is coordinatedin vivo and becomes partially displaced by Ni²⁺ during the nickelchromatographic purification. Because ICP-MS analysis is conducted in aflow of Ar and the most abundant isotope of Ar has the same mass as Ca,Ca binding cannot be determined from the primary mass signal.Nonetheless, because a much higher amount of Ca was present in the cellculture media compared to Zn and yet the ICP-MS signal correspondslargely to zinc, the protein is unlikely to bind calcium. The ICP-MSdata indicate Zn is present in an appreciable amount when PRL-1 ispurified using Ni-IMAC, which suggest that Zn binding occurs in thecell.

Example 4

UV-Vis absorption spectra were used to measure the concentration of thePRL-1 protein. Briefly, the concentrations of purified PRL-1 analogswere measured at 280 nm and calculated using an extinction coefficientof 19420 M⁻¹cm⁻¹. Because of possible metal-protein absorptioninterferences in the high 200 nm region, the validity of the UV-Visquantification method was confirmed by the Bradford method. Spectra ofPRL-1 analogs at concentrations of 10, 5, 2.5, and 1.25 mg/mL werecollected on a Cary 100 UV-Vis spectrophotometer from 800 to 200 nm. Tocorrect for scattering effects in the analysis, PRL-1-WT spectra wereadjusted by subtracting the PRL-1-C170S-C171S spectrum, because it doesnot bind metal. The resulting spectra were resolved and peak positionswere evaluated using GRAMS/AI 7.00 software. Three methods were aimed atmetal displacement. In the first, PRL-1-WT (e.g., wild type) sampleswere reduced using 20 mM β-mercaptoethanol (BME), and imidazole wasadded to a concentration of 10 mM. For 0.5 mM protein samples, thiscorresponds to 400-fold solution excess of BME to compete with the twoC-terminal cysteine thiols and a 200-fold solution excess of imidazoleto compete with the single C-terminal histidine imidazole side chain.New baselines were taken and the spectra recalculated. In the secondmethod, baselines were recalculated for buffer containing 100 μM EDTA,and the spectrum for the PRL-1-WT sample that was exchanged ten millionfold in 100 μM EDTA was recorded. Finally, 10 mg/mL protein samples weredenatured in 5.6 M guanidinium hydrochloride and heated to 90° C., newbaselines were taken, and the spectra were collected at 0, 2, 4 and 6hrs.

Accordingly, characterization of metal coordination by PRL-1 wasdetermined with UV-Vis absorption spectroscopy. The metal coordinationgeometry employed by PRL-1 and its mutants was analyzed using UV-Visabsorption spectroscopy at 800-200 nm. Absorption spectra of PRL-1produced peaks at 280 and 220 nm, which arise from aromatic and peptidebonds within the protein and correspond well with the concentrationdetermined using the Bradford assay. Metal-containing compounds oftenabsorb at several wavelengths in the visible range, and the Ni- andCu-purified PRL-1 proteins also display peaks in the visible range,which correspond to the rust color of the sample. PRL-1 WT shows amaximum at 318 nm (evident as a shoulder on the very large 280 band), abroader signal at 421 nm, and a very broad peak at 526 nm (FIG. 6A).Cu-purified PRL-1 generates a similar spectral profile Ni purified PR1-1(data not shown); however, a small blue shift is observed for theindividual spectral peaks, which is expected when Cu is substituted forNi. As expected, no absorption or visible color was observed when theprotein was purified using resin charged with Zn. This is due to thefact that Zn(II) has a completed d-shell and as such should not showelectronic transitions in the visible spectrum.

Relative to the WT protein, the H166A variant showed the same absorptionprofile, but the peaks were decreased in intensity to a degreeconsistent with the ICP-MS results. The spectra of PRL-1-WT andPRL-1-H166A are distinct from those of the cysteine mutants in thevisible region. At equivalent concentration, individual substitutions atcysteine 170 and/or 171 with serine show complete loss of signal at 421and 526 nm. These mutant proteins were examined at concentrations ashigh as 10 mg/mL (0.5 mM), and no specific absorption bands weredetected. Signal from the WT is evident at 0.1 mg/mL, indicating thatbinding of Ni is decreased by each mutation at least 100-fold.

Deconvolution of the UV-Vis spectra of corrected WT protein wasperformed between 300 and 700 nm and reveals that the 318 nm peak iscomposed of 3 separate absorbance bands at 306, 325, and 372 nm,respectively contributing 54%, 38% and 7% to the total composite peakabsorbance (FIG. 6B). The absorption profile of PRL-1-H166A is highlysimilar to the wild-type protein spectrum. The absence of the imidazolegroup at residue 166 does not shift the absorbance maxima or contributesignificantly to the component bands of the 318 nm absorption, furtherindicating that the imidazole from the histidine within the NGH motifdoes not directly coordinate Ni.

Ni binding in PRL-1 is not affected by reduction of the disulfide bondat PRL-1's active site. Addition of 20 mM BME did not alter the UV orvisible region of the spectral absorption profile, indicating that metalbinding is largely unaffected by reduction of the protein and thepresence of the reducing agent. Subtraction of the reduced spectrum fromthe oxidized confirms that there is no significant difference in metalcoordination between the reduced and oxidized WT samples. To examinewhether a small contribution from a His is made, additional imidazolewas added to the solution to increase the signal strength of a transientassociation. Addition of 10 mM imidazole had no effect on the absorptionprofile, which suggests that access to the Ni is limited. Additionally,no spectral change was observed when the Ni-purified sample was reducedin the presence of imidazole. Ten million-fold exchange of Ni-purifiedWT sample into buffer containing 100 μM EDTA had little effect on thespectrum, and absorbance values for this sample were within error of theWT absorbance. Raising the pH from 7.4 to 8.5 should increase the molarabsorptivity coefficient (E) by approximately 6-fold, but interestingly,no change in ε is observed when the pH is elevated, which may suggestthat the bound Ni is solvent inaccessible in PRL-1. Decreasing the pH to6.5, however, does decrease the intensity of the visible peaks (data notshown).

Ni binding appears to be irreversible at pH 7.4, as displacement of Nifrom Ni-purified PRL-1 requires aggressive measures. When Ni-purifiedPRL-1 was treated with 5.6 M guanidinium hydrochloride at 90° C., thepeaks at 318, 421 and 526 nm only gradually disappear over 6 hrs (datanot shown). The concomitant appearance of a signal at 750 nm wasobserved over the 6 hours, which indicates Ni was released from theprotein and coordinated by water in octahedral geometry. FIG. 6A showsthe electronic absorption spectra of PRL-1 variants purified in thepresence of Ni²′ ion. Three spectra for each protein variant wererecorded from 800 to 200 nm and averaged. Protein concentration for allvariants is 5 mg/mL to within 10% based on A₂₈₀ comparison. Shown arethe spectra from 700 to 300 nm. Visible features are absorbance maximaat 318 and 421 nm. In FIG. 6A, curves are as follows: PRL-1-WT, black;PRL-1-H166A, red; PRL-1-C170S-C171S, orange; PRL-1-C1705, green;PRL-1-C171S, blue.

FIG. 6B shows a resolved spectrum of PRL-1 WT. Resolution of the UV-Visspectrum of Ni-purified PRL-1 WT protein between 300 and 700 nm fits thedata using 5 curves. The 318 nm shoulder is resolved into peaks at 306,325, and 372 nm. The sum of the resolved absorbance peaks (gray dashedline) and the raw data (solid black line) are both shown. The peaks at421 and 526 nm are also present.

The inset of FIG. 6B shows the spectra from 600 to 400 nm are shown atan expanded scale for visualization of absorption maxima at 421 and 526nm.

Example 5

Electrospray ionization mass spectrometry (ESI-MS) was used to analyzethe samples. Briefly, ESI spectra were acquired on a Q-T of-2 (MicromassLtd, Manchester UK) Hybrid Mass Spectrometer operated in MS mode andacquiring data with the time of flight analyzer de-tuned to 8000resolution (FWHH) for sensitivity. For whole protein ESI spectra,purified protein samples were diluted to 1 μg/μL in untreated(“oxidizing”) 20 mM ammonium bicarbonate, pH 8.2 and allowed to incubateat room temperature. Time points were taken on the first, fourth, andseventh day post-purification and frozen at −80° C. until ready for use.Samples were desalted on a short (3 cm×1 mm I.D.) reverse-phase (RP)HPLC column (Hamilton PRP1, Reno, Nev.). Samples were loaded onto thecolumn from a 1% formic acid solution with protein (5 μg), washed insame solution, and eluted with 90% MeOH/0.5% formic acid directly intothe ESI source. The cone voltage was 60 eV, and the voltage on thecollision cell was 20 V. Spectra were acquired over the mass range 800to 3000 u, accumulating data for 5 seconds per cycle.

For tryptic digests, PRL-1-WT, PRL-1-H166A and PRL-1-C170S-C171S werediluted to 1 mg/mL in ammonium bicarbonate, pH 8.2, with or without 20mM β-mercaptoethanol, and trypsin (Promega) was added in a 1:50 ratioprotease:protein (w/w) and incubated at 37° C. for 16 hrs. To confirmcomplete digestion, samples at various time points were run on SDS-PAGEand visualized by silver staining Coupled to MS analysis, capillary HPLCseparations were performed using a Zorbax SBC18 RP column (5 cm×0.32 mmI.D., 3.5 μm bead size, 300 Å pore size) packed by Micro-Tech Scientific(Sunnyvale, Calif.), with a chromatograph (Waters capLC XL, Milford,Mass.) that develops gradients at 10 μL/min. A linear gradient of 20 to80% B was applied over 120 minutes. The solvents were: A 99% H₂O, 1%MeOH, B 99% MeOH, 1% H₂O, both 0.08% formic acid. Argon was admitted tothe collision cell at a pressure that attenuates the beam to about 20%.This corresponds to 16 psi on the supply regulator or 5.3×10⁻⁵ mBar on apenning gauge near the collision cell. The collision cell was operatedat 8 V for maximum transmission, and spectra were acquired over therange 250 to 2000 u, accumulating data for 8 seconds per cycle.

NMR experiments were conducted to analyze the samples. Briefly, 2D¹H-¹⁵N HSQC spectra were acquired on a Bruker Avance 800 MHz NMRspectrometer using a cryogenic, triple-resonance probe equipped withpulse field gradients. Water suppression was accomplished usingflip-back pulses. All spectra were obtained at 37° C. and acquired in 16scans with 2048 points in ¹H. 256 or 128 increments were collected in¹⁵N for WT and C170S-C171S, respectively. Samples were prepared in 50 mMsodium phosphate, 100 mM NaCl at pH 6.5 and contained 5% D₂O. Theconcentration of WT PRL-1 and PRL-1-C170S-C171S was 1.0 mM and 0.8 mM,respectively. Sample reduction was accomplished by addition of 10 mM DTTat least 24 hours prior to spectral acquisition. ¹H chemical shifts werereferenced with respect to an external DSS standard in D₂O. Indirectreferencing relative to ¹H was determined for ¹³C and ¹⁵N, assumingratios ¹³C/¹H=0.251449530 and ¹⁵N/¹H=0.101329118. Computer programsnmrPipe and Sparky were used for data processing and spectral analysis.

Example 6

To confirm that the MAP tag motif is entirely responsible for metalbinding, we synthesized NCC. The NCC peptide was synthesized andpurified, and the peptide was incubated with metal-charged IMAC resin.Complex formation was verified using electrospray ionization massspectrometry (ESI-MS) operating in negative ion mode (FIG. 3).

In the NCC peptide, the absorption signal mimics that observed for thepeptide-metal complex embedded in PRL-1, but the complex is more exposedin the tripeptide. The absorption in the visible range intensifies whenthe pH is raised from 6 to 7.4. Acidification diminishes the signalintensity and when returned to the more basic pH, full signal fails tobe restored, indicating irreversible loss of some metal from the squareplanar geometry may occur. Elevation of the pH to 10 suggests a fifthligand, presumably water or hydroxide, is coordinated, generating asquare pyramidal arrangement. At pH 7.4, this ligand is presumablyhighly exchangeable and too transient to be detected by absorptionspectroscopy. The exchangeable ligand provides an avenue forparamagnetic relaxation of the bulk solvent and can be detected by MRI(FIG. 4). Additionally, the metal was added by mixing the metal saltinto aqueous solution containing the peptide. Depending on theconditions, the absorption spectrum of the peptide either paralleled thecomplex formed via metal transfer or was distinctly different from thatobtained for the full-length protein. The differing spectrum revealsthat the metal may be coordinated solely by sulfur in this case.Although the NCC peptide is sufficient to confer metal binding, the typeof complex generated depends on the method by which the metal isintroduced to the peptide. Transmetallation provides an efficientpathway for incorporation of the metal into the NCC tripeptide togenerate the extremely high affinity square planar complex.

The GGH-like metal complex of NGH is shown in FIG. 1, Structure 1. Weenvisioned two possible chemical structures for the metal-bound NCCcomplex, which are shown in FIG. 1, Structure 2 and Structure 3.Structure 2 corresponds to a GGH-like arrangement that involvescoordination of the metal by several deprotonated backbone N atoms. Inthis case, the imidazolium nitrogen from His would be replaced in NCCwith the sulfur from the Cys in the third position in the coordinatedcomplex. This complex would have 3N:1S coordination, which is notconsistent with our absorption spectra. To verify the unique, specificcoordination by NCC, NGC also was synthesized and confirmed theimportance of the central Cys side chain in metal binding. The NGCpeptide incubated with the IMAC resin does not produce an absorbancespectrum that resembles the metal-bound NCC or PRL-1 spectrum. As such,we concluded that GGH-like coordination is not utilized and must have amuch lower affinity for binding metal in the specified geometry.

Structure 3 is completely in line with the data obtained for both thepeptide alone and the peptide in the context of the larger protein. Theabsorption spectrum of both indicate that two sulfur atoms participatein the complex along with two nitrogen atoms, one which is deprotonatedand the other which has a single proton attached. Structure 3 may notinvolve the N-terminal amine group, which would likely cause differencesin the spectra of the peptide and protein because this atom would beembedded in a peptide bond in the protein. Based in part on thisstructure, the nitrogen from Q, H, K, or R may be substitute for N inthe first position of the motif.

The metal binding site and coordination geometry was studied. Thefinding that PRL-1 binds divalent metal cations is interesting becausespecific metal coordination by other PTPases has not been reported. TheC-terminal sequence of PRL-1 is unique among this enzyme family, and itencodes both a GGH-like motif, which in certain cases is able to bindmetal, but not in this case, and the novel MAP tag motif The geometry ofGGH-like motifs is square planar. The characteristic rust color observedwith the concentrated PRL-1 samples parallels the color of the GGHpeptides, which suggests that Ni coordination occurs in a square planargeometry. Our studies further reveal that Ni is bound in the C-terminusof PRL-1. Metal coordination is not accomplished by the GGH-like motifin PRL-1 because mutation of the would-be essential His has littleaffect on binding. Despite the fact that both tripeptides bind usingsquare planar coordination, the chemistry for each is distinct.

UV-Vis spectra derived from Ni-purified WT PRL-1 display bands near 318,421 and 526 nm. For peptides and organic molecules binding Ni in asquare planar geometry via nitrogen ligands, characteristic d-d bandsnear 420 nm are reported frequently in the literature. Maroney et al.(“Theoretical Study of the Oxidation of Nickel Thiolate Complexes byO(2).” Inorg Chem. 1996 Feb. 14; 35(4):1073-1076) performed ab initiocalculations and made spectral assignments of synthetic peptide mimeticsthat coordinate Ni(II) and Cu(II) using square planar geometry. With theexception of a single band, their observations and calculations are inclose agreement with the observed spectrum from PRL-1. Separately, thereduced, square-planar form of NiSOD was analyzed using densityfunctional theory, the results of which show similar relevant features.The locations of the bands as well as the trend in their relativeintensities in these systems are well preserved in the PRL-1 proteinspectrum. Based on these and other studies, it is likely the 526 nm bandarises from Ni_(xy)-->Ni_(x2-y2) transitions. Similarly, the 421 nm bandmay come from Ni_(z2)-->Ni_(x2-y2) transitions. The strong 318 nm bandreflects the ligand-metal charge transfer band (LMCT). Resolution of thespectrum reveals that this absorbance is likely a composite of 3 bandsat 306, 325, and 372 nm. Using the other structures as a guide, thesharpest, most intense band at 306 nm would correspond to transitionsfrom sulfur atoms, with the other two bands likely arising from nitrogendonation or possibly weaker sulfur donation.

Within the series of C-terminal mutants, only mutations involving C170and C171 abolished signal at these wavelengths. Most notably, the 318 nmenvelope corresponding to the LMCT is lacking in the cysteine mutantspectra. With respect to the entire absorption profile, the small verybroad absorbance in the low 300 nm region for the cysteine mutantsappears to be an artifact from high-intensity bands at lower wavelengthsor a scattering effect, as it is positioned incorrectly to be one of the318 nm component bands. The bands attributable to transitions within Nid-orbitals (421 nm, 526 nm) also are absent when sulfur is substitutedby oxygen. The original bands are absent and no additional bands appearupon mutation of either C170 or C171 in the absorption spectra. Also nodisulfide bonds involving these residues are observed in the MS data. Assuch, it seems that both cysteines directly participate in metalcoordination. Our ICP-MS data further indicate that both sulfur atomsfrom C170 and C171 are required for tight Ni-binding, as neither sulfuratom alone appears to be sufficient.

The spectral features generated by PRL-1 also suggest Ni is coordinatedby nitrogen atoms in the remaining two positions. Frequently imidazoliumN from His or backbone amide N are observed to coordinate metals such asZn, Cu and Ni. In model peptides and proteins that bind Ni using squareplanar geometry, some of the ligating nitrogen groups becomedeprotonated, and the negatively charged species binds the metal. Theabsorption band at 526 nm indicates that PRL-1 coordinates Ni via adeprotonated nitrogen, whereas the 421 nm absorption generated in thePRL-1 spectrum reflects coordination by a singly protonated nitrogen.Mutations of the His in the GGH-like motif to make PRL-1-H166A revealedthat this histidine's side chain does not substantially influence Nibinding. PRL-1 encodes three other His in addition to H166. H23, H64 andH103 are located in distant regions of the sequence and are unlikely toremain tightly associated under denaturing conditions. Attempts tounfold the protein so as to release the metal required vigoroustreatment with high concentrations of denaturants at high temperaturefor several hours. As chemical and heat denaturation permit only slowrelease of Ni and CD data show that PRL-1 completely unfolds well beforeNi is released, the metal is likely coordinated by atoms clustered in ashort segment of the protein.

The GGH-type peptides achieve extremely tight binding(K_(d)˜10⁻¹⁷-10⁻¹⁸), which is accomplished in part by deprotonatedbackbone amide nitrogens within the motif. In light of the extremelytight binding observed for PRL-1 and the predicted contribution fromnitrogen atoms to Ni ligation, backbone amides from PRL-1 mayparticipate in metal coordination. Because the absence of eithercysteine abolishes binding and truncation removes both the side chainand the backbone amide, testing the hypothesis that metallation involvesthe amide nitrogens adjacent to C170 and C171 using mutagenesis is notfeasible. Structural studies are the best way to identify the additionalcoordinating atoms, and these studies are underway in our laboratory.

Example 7

Cu-NCC at physiological pH was placed in a tube surrounded by salinebags and imaged using a standard MRI at the Hoglund Brain Imaging Centerat the University of Kansas Medical Center (FIG. 4). In FIG. 4, thebright signal in the center corresponds to the Cu-NCC complex, whereasthe less bright signal corresponds to the saline solutions. Blackcorresponds to the tube and air.

Therefore, the present invention is well adapted to attain the ends andadvantages mentioned as well as those that are inherent therein. Thepresent invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope. All references recitedherein are incorporated herein by specific reference.

1-20. (canceled)
 21. A composition comprising: a peptide including thesequence XC₁C₂; wherein X is any non-cysteine amino acid; and wherein C₁and C₂ are the same or different; and wherein C₁ and C₂ individually arechosen from a cysteine, a sulfur-containing alpha or beta amino acid,and a cysteine-like nonnatural amino acid; and a metal atom or ion boundto the sequence XC₁C₂ such that the metal atom or ion is bound with adissociation constant at least as high as when bound in square planar orsquare pyramidal orientation.
 22. The composition of claim 21, whereinsaid metal atom or ion is bound with greater than 90% retention for 6hours.
 23. The composition of claim 21, wherein said metal atom or ionis bound with greater than 90% retention after treatment with hightemperature.
 24. The composition of claim 23, wherein said hightemperature is about 90° C. for less than 6 hours.
 25. The compositionof claim 21, wherein said metal atom or ion is bound with greater than90% retention upon treatment with aqueous high salt conditions.
 26. Thecomposition of claim 25, wherein said aqueous high salt conditions areabout 5.6M guanidinium hydrochloride for less than 6 hours.
 27. Thecomposition of claim 21, wherein said metal atom or ion is bound withgreater than 90% retention upon treatment with strong acid.
 28. Thecomposition of claim 21, wherein said metal atom or ion is boundessentially irreversibly at pH 7.4.
 29. The composition of claim 21,wherein said metal atom or ion is bound with a dissociation constantwhich is greater than the dissociation constant of the metal atom or ionwith the sequence GGH.
 30. The composition of claim 21, wherein saidmetal atom or ion is bound only within the sequence XC₁C₂.
 31. Thecomposition of claim 21, wherein X is chosen from asparagine, glutamine,histidine, lysine, and arginine.
 32. The composition of claim 21,wherein C₁ and C₂ are each cysteine.
 33. The composition of claim 21,wherein said metal atom or ion is selected from a Group 3 metal, a Group5 metal, a Group 6 metal, a Group 7 metal, a Group 8 metal, a Group 9metal, a Group 10 metal, a Group 11 metal, a Group 12 metal, a Group 13metal, a Group 14 metal, and a Group 15 metal.
 34. The composition ofclaim 33, wherein said metal atom or ion is selected from a Group 5,Group 10, Group 11, and Group 12 metal.
 35. The composition of claim 34,wherein said metal atom or ion is a Group 5 metal.
 36. The compositionof claim 35, wherein said Group 5 metal is vanadium.
 37. The compositionof claim 34, wherein said metal atom or ion is a Group 10 metal.
 38. Thecomposition of claim 37, wherein said Group 10 metal is selected fromthe group consisting of Ni, Pd, and Pt.
 39. The composition of claim 34,wherein said metal atom or ion is a Group 11 metal.
 40. The compositionof claim 39, wherein said Group 11 metal is selected from the groupconsisting of Cu, Ag, and Au.
 41. The composition of claim 40, whereinthe metal atom or ion is Cu.
 42. The composition of claim 34, whereinsaid metal atom or ion is a Group 12 metal.
 43. The composition of claim42, wherein said Group 12 metal is selected from the group consisting ofZn, Cd, and Hg.
 44. The composition of claim 21, wherein said peptide isisolated and purified.
 45. The composition of claim 21, wherein saidpeptide is linked to a protein, a polypeptide, an antibody, a growthfactor, or a carbohydrate.
 46. The composition of claim 45, wherein saidpeptide is linked to an antibody.
 47. The composition of claim 21,wherein said peptide is included in the sequence of a polypeptide orprotein.
 48. The composition of claim 21, wherein said composition isselected from the group consisting of imaging compositions andtherapeutic compositions.